CN116396370A - 具有提高镉抗性及促进生物量提升的植物编码基因及蛋白和转基因株系 - Google Patents
具有提高镉抗性及促进生物量提升的植物编码基因及蛋白和转基因株系 Download PDFInfo
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Abstract
本发明公开了具有提高镉抗性及促进生物量提升的植物编码基因及蛋白和转基因株系。本发明通过对镉胁迫处理柳枝稷开展RNA‑seq测序,筛选出上调倍数最高的差异表达基因,基因组注释表明该基因编码热激蛋白70家族(Hsp70)成员——分子伴侣BiP(Binding Protein)蛋白,将该基因命名为PvBiP2。构建其过表达载体转入镉敏感型酵母突变体菌株(ycf1)中,在含镉培养基上转基因酵母的生长状况明显优于对照组。将所述PvBiP2基因在柳枝稷中过表达,发现可以显著提高转基因柳枝稷的镉抗性并促进柳枝稷生物量提高。
Description
技术领域
本发明涉及基因及蛋白,特别涉及具有提高镉抗性及促进生物量提升的植物编码基因及蛋白。
背景技术
由于经济发展较快,工业化、城市化进程造成大量“三废”排放以及农业生产中农药、化肥的滥用,土壤重金属镉(Cd)污染程度呈直线上升趋势,远高于其他污染物超标率,高居土壤重金属污染榜首,土壤Cd污染治理迫在眉睫、刻不容缓。
柳枝稷(Panicum virgatum L.)是起源于北美的多年生高杆C4草本植物,与传统作物相比,柳枝稷的适应性强,抗虫、需肥少,产量高,最高产量可达到74.1t·hm2。柳枝稷不仅生物量大,而且细胞壁干物质木质素和纤维素的含量极高,是生产生物热能、乙醇和甲烷的模式植物,早在1992年柳枝稷就被美国能源署(DOE)选择确立为模式生物能源作物。作为主要的重金属污染物,镉(Cd)能被作物从土壤环境里吸收,转运到叶和籽粒这样的食用部分,同时这些粮食中的Cd能造成人类患上严重的、长期的疾病。对Cd污染土地种植覆盖植被是有效的治理策略。多年生的生物能源草,一般具有较高生物量、拥有强健发达根系,是Cd污染地域重新种植的合适候选植物,能用来还原地面覆盖植被甚至逐渐恢复生态系统。柳枝稷很适合边际土地上种植,它强大的根系能有效地改善土壤流失,重复种植收获柳枝稷(生物量),能吸收、减少土壤中Cd含量。经燃烧或细胞发酵之后的灰烬(残渣)被收集(利用),不会带来环境污染的风险。研究表明,柳枝稷对Cd有一定耐性,但Cd对柳枝稷生长的抑制作用也很明显。是否可从柳枝稷中挖掘Cd胁迫响应基因,对实施柳枝稷分子遗传改良,创新能源草-Cd修复兼用型柳枝稷种质材料,为治理修复Cd等重金属污染土地、恢复生态功能具有重要意义。
发明内容
发明目的:本发明目的是提供具有提高镉抗性及促进生物量提升的植物编码基因,将该基因命名为PvBiP2。
本发明的另一目的是提供具有提高镉抗性及促进生物量提升的植物编码的蛋白和转基因株系。
技术方案:所述具有提高镉抗性及促进生物量提升的植物编码基因,CDS序列如seq.id.no.1所示。
所述基因编码的蛋白,序列如seq.id.no.2所示。
本发明通过对镉胁迫处理柳枝稷开展RNA-seq测序,筛选出上调倍数最高的差异表达基因,基因组注释表明该基因编码热激蛋白70家族(Hsp70)成员——分子伴侣BiP(Binding Protein)蛋白,将该基因命名为PvBiP2。构建其过表达载体转入镉敏感型酵母突变体菌株(ycf1)中,在含镉培养基上转基因酵母的生长状况明显优于对照组。将所述PvBiP2基因在柳枝稷中过表达,发现可以显著提高转基因柳枝稷的镉抗性并促进柳枝稷生物量提高。
一种由前述基因构建的pCambia1305.2-PvBiP2转基因株系。
有益效果:本发明与现有技术相比,具有如下优势:
1、本发明提供了一种具有提高镉抗性及促进生物量提升的植物编码基因PvBiP2,该基因目前尚未见相关的报道研究,该基因源自于镉胁迫处理柳枝稷根系RNA-seq测序结果中的上调差异表达基因;通过构建PvBiP2的酵母表达载体,利用酵母点板实验可验证本发明所述基因能提高酵母镉抗性。
2、本发明将PvBiP2基因柳枝稷中过表达,发现该基因可以显著提高转基因柳枝稷镉抗性,镉胁迫处理后,转基因植株的表型、生物量以及生理和光合指标都明显优于WT,并且能够在根系维持较高浓度镉、地上部分较低浓度镉含量。与野生型(WT)柳枝稷比较,正常条件下,转基因植株的株高、茎粗和叶片宽度显著(P<0.05)高于WT。
3、本发明所述的PvBiP2基因为培育能源草-镉修复兼用型柳枝稷种质提供基因资源,可应用于镉污染地区的治理中,在研究植物生物修复技术、改善土壤重金属污染状况等方面起着重要作用。
附图说明
图1为PCR扩增的PvBiP2基因的产物条带;
图2为PvBiP2基因在镉胁迫下的酵母功能验证;
YCF1/pGAD426-GUS为空白对照Cd敏感酵母菌株,YCF1/pGAD426-PvBiP2为转基因酵母菌株;
图3为过表达载体pCambia1305.2-PvBiP2构建与转化农杆菌,a.PvBiP2连接到pEND-linker克隆载体;b.线性化后,pEND-PvBiP2经LR反应连入pCambia1305.2双元表达载体;c.农杆菌单菌落(1-3)菌液PCR鉴定;
图4为农杆菌介导过表达PvBiP2柳枝稷遗传转化流程,a.胚性愈伤组织b.筛选抗性愈伤组织c.抗性芽分化d.抗性芽生根e.转基因植株f.转基因植株温室培养;
图5为转PvBiP2基因柳枝稷鉴定与表达量分析,a.PvBiP2基因过表达载体模式;b.PCR扩增hpt II基因(M:maker;EV:pCambia1302-PvBiP2质粒:WT:野生型柳枝稷;1-5:5个转基因株系);c.柳枝稷转基因株系GUS染色结果;d.野生型和转基因柳枝稷PvBiP2基因表达量比较(野生型的表达量设为1,误差线表示平均值±SE,n=3,不同小写字母表示差异显著水平为P<0.05);
图6PvBiP2-OE柳枝稷和野生型柳枝稷在无Cd和Cd胁迫下表型和生物量分析PvBiP2-OE和野生型柳枝稷在无Cd和Cd胁迫处理30d后的茎叶(A)和根系(B)表型分析;不同浓度镉处理下柳枝稷PvBiP2-OE株系和野生型的茎叶鲜重(C)、茎叶干重(D)、根系鲜重(E)和根系干重(F)分析,误差线表示平均值±SE,n=4,不同小写字母表示差异显著水平为P<0.05);
图7为PvBiP2-OE柳枝稷和野生型柳枝稷在无Cd和有Cd胁迫处理下生理指标分析,
PvBiP2-OE和野生型柳枝稷在无Cd和Cd胁迫处理30d后测量如下生理指标:(A)叶片相对含水量(RWC);(B)谷胱甘肽(GSH);(C)相对电导率(EL);(D)丙二醛(MDA)含量;(E)脯氨酸含量;(F)可溶性蛋白含量,误差线表示平均值±SE,n=4,不同小写字母表示差异显著水平为P<0.05;
图8为PvBiP2-OE柳枝稷和野生型柳枝稷在无Cd和有Cd胁迫处理下光合作用相关指标分析,PvBiP2-OE和野生型柳枝稷在无Cd和Cd胁迫处理30d后测量光合生理指标:(A)叶绿素含量;(B)净光合速率(Pn);(C)胞间二氧化碳浓度;(D)气孔导度(Cond);(E)蒸腾速率(Tr);(F)水分利用效率(WUE),误差线表示平均值±SE,n=4,不同小写字母表示差异显著水平为P<0.05;
图9为过表达PvBiP2对柳枝稷的Cd吸收和迁移的影响,PvBiP2-OE和野生型柳枝稷在无Cd和Cd胁迫处理30d后测量Cd含量:(A)茎叶中Cd浓度;(B)根系中Cd浓度;(C)植株中Cd含量;(D)Cd迁移系数,误差线表示平均值±SE,n=4,不同小写字母表示差异显著水平为P<0.05。
具体实施方式
本实施例是实验材料和试剂:
(1)菌株与载体:酵母菌株YCF1、G19、HOG1以及入门载体pEND-linker和双元植物表达载体pCambia1305.2均购自invitrogen公司。大肠杆菌DH5α购自康为世纪生物科技有限公司。镉敏感型酵母突变体菌株(ycf1)购自euroscarf公司。
(2)酶与试剂盒:实时荧光定量PCR试剂盒Roche SYBR GREEN Master(ROX)购自罗氏(Roche)公司,T4链接酶、限制性内切酶购自NEB公司,植物基因组DNA试剂盒购自原平皓(天津)生物技术有限公司,植物RNA试剂盒、质粒提取试剂盒、PCR产物回收试剂盒、琼脂糖凝胶DNA回收试剂盒均购自OMEGA公司,氨苄青霉素(Amp)、卡那霉素(Kan)、利福平(Rif)、潮霉素B(HygB)等购自Sigma公司,其他化学试剂与耗材统一购自南京寿德器材有限公司。
(3)反应引物:由生工生物工程(上海)股份有限公司合成。
实施例1
PvBiP基因的克隆
(1)PCR扩增获得目的基因片段
根据PvBiP2基因编码序列,设计合成引物,以gDNA为模板,通过Q5高保真DNA聚合酶扩增PCR扩增获得目的基因片段,反应体系如下。PCR反应程序为:98℃预变性3min;98℃变性30s,63℃退火10s,72℃延伸1 min,30个循环;72℃延伸5min;10℃,10min。
(2)PCR产物回收
PCR产物回收采用E.Z.N.A Gel Extraction Kit-Spin试剂盒回收。方法为:
①将含有目的片段的胶块切下,放入1.5ml离心管中,加入等体积的BindingBuffer。
②金属浴调至60℃,将离心管放在金属浴上7-10min,每2-3min上下颠倒离心管,使胶块充分溶解。
③将吸附柱放于2ml的收集管中。
④当胶块完全溶解后,将全部液体转移到吸附性注中,10000g,1min,倒掉废液,将吸附柱放回收集管中。如果液体过多,可以分次转移,直至所有液体都转移到吸附柱中。
⑤加入300ul Binding Buffer于离心柱中,13000g离心1min,倒掉上清,将吸附柱放回收集管中。
⑥在SPW Wash Buffer中加入适量体积的无水乙醇,往吸附柱中加入700ul SPWWash Buffe,13000g离心1min,倒掉废液,将吸附柱放回收集管中。
⑦重复步骤⑥。
⑧将倒掉废液的收集管重新放回离心机,13000g离心2min,将吸附柱放到新的离心管中,在空气中放置2-3min。
⑨加入30ul Elution Buffer(已经65℃预热),室温静置3min,13000g离心1 min.
⑩使用酶标仪测量回收DNA浓度,置于-20℃冰箱保存。
通过PCR扩增得到PvBiP2全长,扩增产物结果见图1。
(3)目的基因片段与入门载体pEND-Linker连接
将双酶切后的入门载体pEND-linker与PvBiP2目的基因片段连接,反应体系如下:
将上述液体混匀,16℃,反应2-3h。
(4)连接产物转化大肠杆菌感受态细胞DH5α:
①取冰盒,从-80℃超低温冰箱取出大肠杆菌感受态细胞置于冰上。
②在感受态刚刚解冻时加入10ul连接产物,冰上放置30min。
③打开金属浴,温度调至42℃,42℃热激1 min,冰上放置2-3min。
将感受态加入1 mL LB液体培养基(无抗生素),37℃,180rpm活化30min。
④室温7000rpm,离心1min,于超净工作台中倒掉上清,留少许重悬沉淀,把重悬的菌液涂布在抗性培养基上,放入37℃培养箱培养一夜。
⑤挑选3-5个单克隆,PCR验证。
转化大肠杆菌感受态细胞挑取阳性克隆PCR检测,PCR检测反应体系如下:
PCR产物进行跑胶检测,选取与目标条带大小一致菌液提取质粒,送南京擎科生物科技有限公司测序。
实施例2
酵母异源表达与耐镉性分析
(1)酵母表达载体构建
提取测序结果正确的阳性克隆质粒,具体方法如下:
①收集菌体,收集1-5ml的大肠杆菌液体,室温10000g,离心1min倒掉废液,收集菌体。
②加入250μL Solution I(按要求加入适量体积的RNaseA),涡旋1min,充分裂解菌体。
③加入250μL Solution II,缓慢的上下颠倒8-10次。
④加入250μL Solution III,立即混匀,上下颠倒8-10次,室温13000g,离心10min。
⑤将吸附柱插入收集管中,将上清转移到收集管中(如果一次未转移完可分次转移),室温13000g,离心1min,去掉废液,将吸附柱放回收集管中。
⑥加入500μL HBC Buffer,室温13000g,离心1min,去掉废液,将吸附柱放回收集管中。
⑦加入700μL DNA Wash Buffer,室温13000g,离心1min,去掉废液,将吸附柱放回收集管中。
⑧重复步骤⑦。
⑨将吸附柱放回空收集管中,室温13000g,离心2min,将吸附柱放入新的离心管中,在空气中晾2-3min。
⑩在吸附膜中间位置加入60μL Elution Buffer,孵育2-3min,室温13000g,离心1min。
⑩使用酶标仪检测浓度,于-20℃冰箱保存。
用限制性内切酶Pvu I线性化处理,体系如下:
反应条件:37℃,酶切1h。凝胶电泳检测,并切胶回收目的片段。将目的片段与表达载体pGAD426进行LR重组反应,反应体系如下:
反应条件:25℃,1h。转化大肠杆菌感受态DH5α,挑取阳性克隆,通过载体大小和通过PCR检测,确定重组反应得到正确的表达载体并提取阳性克隆质粒。
(2)YCF1(镉敏感酵母菌株)感受态细胞感受态细胞制备
①从实验室-80℃冰箱中划取YCF1酵母细胞于YPDA培养基中,30℃培养箱中培养1-2d。
②挑单克隆于提前配制好的YPDA培养液中,放置在28℃,210rpm摇床上过夜。
③将摇浓后的菌体转大摇,放在28℃,210rpm摇床中,当OD600=0.8-1.0时,取出菌体待用。
④转移上述菌体,4℃,500g,4min离心,丢弃上清。。
⑤加入10ml EZ1 solution重悬菌体,4℃,500g,4min离心,丢弃上清。
⑥加入1ml EZ2 solution重悬菌体,分装感受态,每个样品体积为10μl。纸巾包裹放入-80℃冰箱中保存。
(3)镉敏感酵母YCF1转化步骤
①取制备的YCF1感受态,2-3min后,感受态初解冻时,在无菌环境中向感受态细胞中加入(0.2-1μg)pGAD426-PvBiP1a质粒,轻轻混匀。
②向上述液体感受态细胞中加入100μl EZ3 solution,立即混匀。
③将混合均匀的液体放置在30℃培养箱中培养60-90min,在培养过程中每20min混匀液体一次,整个过程混匀2-3次。
④培养结束后将液体均匀地涂抹在二缺酵母培养基(SD/-Ura)上,30℃恒温培养2-3d。
(4)酵母菌株耐镉性检测
①使用牙签挑取YCF1单一克隆接种于1mL一缺液体培养基(SD/-His-Ura)生长,28℃,210rpm震荡2-3d
②待菌液生长至平台期,使用无菌水按梯度依次稀释10倍浓度
③取5μL按照浓度依次点在100μmol·L-1CdCl2的一缺培养基(SD/-Ura)中30℃恒温培养5-6d,观察酵母表型,采集照片。
在100μmol·L-1CdCl2 SD/-Ura选择培养基平板上,与对照pGAD426-GUS菌株比较,PvBiP2过表达酵母菌株的生长显著增强(图2),呈现出一定耐Cd表型。
实施例3
柳枝稷PvBiP2遗传转化及PvBiP2-OE株系的鉴定
(1)植物过表达载体构建
提取获得的PvBiP2阳性克隆质粒,经线性化后与植物表达载体pCambia 1305.2进行LR重组反应,转化大肠杆菌,PCR检测。质粒转化农杆菌菌株AGL1,获得含有正确表达载体的农杆菌克隆,将农杆菌菌液加甘油冻存备用。具体方法同实例2中。重组载体pCambia1305.2-PvBiP2以潮霉素磷酸转移酶基因(hptII)为选择标记,目的基因在玉米泛素启动子UbiP的驱动下控制下表达。通过PCR扩增PvBiP2基因全长,并与pEND-linker入门载体连接,转化大肠杆菌,提取质粒PCR检测阳性克隆(图3a)。线性化质粒后与双元表达载体pCambia1305.2进行同源重组反应连接,成功构建柳枝稷过表达载体pCambia1305.2-PvBiP2(图3b)。将重组载体pCambia1305.2-PvBiP2导入农杆菌AGL,选取单菌落进行PCR鉴定(图3c)。编号1、2、3的PvBiP2基因为明显的单一条带,大小正确,扩增效率高,均能够用于后续遗传转化。
(2)柳枝稷遗传转化及抗性株系再生
当年收获的柳枝稷“HR8”种子经烘干、50%硫酸软化种皮,再经2.5%次氯酸钠消毒、无菌水清洗后均匀接入愈伤组织诱导培养基,暗培养约1月诱导出愈伤组织(图4a),多次继代后将胚性愈伤组织(图4b)挑选出用于于遗传转化,经农杆菌侵染和共培养后,将愈伤组织转移至含有375mg·L-1Augmentin和50mg·L-1Hygromycin B的愈伤组织筛选培养基,约4周左右,大部分愈伤组织逐渐褐变死亡,只有少数愈伤组织正常生长。筛选后将生长状态良好的愈伤组织转移到柳枝稷分化培养基上光照培养继续生长,约一个月后部分抗性愈伤组织分化出芽(图4c)并逐渐长成抗性苗(图4d)。每丛愈伤组织只选取一株苗作为一个转基因株系继续扩繁。将各株系分化出的高度达到3cm以上的幼苗转移到组培瓶中继续生根培养(图4e),待根系长度达到5cm左右移植到花盆土培(图4f)进行后续研究。
(3)柳枝稷转基因株系的鉴定
使用构建的双元表达载体pCambia1302-PvBiP2质粒,转入农杆菌,侵染柳枝稷愈伤组织,经过共培养、抗压筛选、分化、生根等阶段,获得pCambia1305.2-PvBiP2转基因株系。根据载体组成,pCambia1305.2载体中含有GUS报告基因和潮霉素磷酸转移酶基因hptII(图5A),因此采取GUS染色分析和hptII基因PCR扩增来对转基因株系进行鉴定。经GUS染色,5个株系叶片出现深浅和分布不一的GUS报告基因表达的蓝色效果,WT中无此效果(图5C),同时这5个株系均扩增出了hptII基因条带(图5B,表明这5个柳枝稷转基因株系中表达载体已经整合进基因组,是阳性转基因株系,将它们命名为line1-line5。经qRT-PCR检测line1-line 5转基因株系的相对表达量(图5D),line 1和line 5中PvBiP2相对表达量显著高于其他株系和野生型(WT)(P<0.05),所以选取这两个株系为后续耐镉性实验材料。
实施例3:
过表达PvBiP2基因提升了柳枝稷生物量和镉耐性
(1)过表达PvBiP2基因促进柳枝稷生长
将T0代转基因株系与同批次非转基因WT组培苗在移栽土培90d后,统计株高、茎粗、叶宽、节间长、单株分蘖数等生长指标(见下表),PvBiP2-OE株系上述指标均显著高于WT植株(P<0.05)。在R2期收苗后,PvBiP2-OE株系line 1和line 5茎叶鲜重分别比野生型高出16.0%和23.2%,干重则是高出20.9%和24.4%(图6C&E),差异显著(P<0.05)。PvBiP2过表达促进了转基因植株的生长与生物量增加。
柳枝稷过表达PvBiP2株系和野生型的生理和生长性状表
(2)过表达PvBiP2能缓解柳枝稷生长时的镉胁迫
镉胁迫处理时,经30d镉处理的柳枝稷PvBiP2-OE株系和WT都表现出叶片黄化,根系和茎叶生长下降的现象。但是,PvBiP2-OE株系保有更多绿叶和有活力根系(图6A&B);100μmol·L-1CdCl2处理下PvBiP2-OE line 1和line 5株系茎叶干重生物量比WT分别高45.4%和44.6%,根系生物量高83.7%和61.2%(图6D&F)。因此,过表达PvBiP2能缓解了柳枝稷生长时的镉胁迫。
(3)镉胁迫下过表达PvBiP2柳枝稷生理和光合指标优于WT
镉胁迫处理后对柳枝稷PvBiP2-OE株系和WT的生理指标进行测定:在50~100μmol·L-1CdCl2处理下,PvBiP2-OE株系比WT其叶片含水量(RWC,图7A)显著升高(P<0.05),电导率(EL,图7C))和丙二醛(MDA,图7D)显著降低,表明PvBiP2-OE株系有更好的水分状态和膜系统完整性。值得注意,在WT中谷胱甘肽(GSH,图7B)含量显著高于PvBiP2-OE株系中。虽然镉胁迫还引起PvBiP2-OE株系和WT中脯氨酸(图7E)和可溶性蛋白含量下降(图7F),但PvBiP2-OE株系中要显著高于WT。对光合指标进行测定,50~100μmol·L-1CdCl2处理下,柳枝稷PvBiP2-OE株系和WT的叶绿素含量(Chl,图8A)、净光合速率(Pn,图8B)、蒸腾速率(Tr,图8C)、气孔导度(Cond,图8D)都受到抑制,但转基因株系的抑制率显著低于WT,而且无镉条件下转基因株系叶绿素含量、净光合速率(Pn)显示出明显更高(P<0.05)。
(4)过表达PvBiP2柳枝稷增加从土壤镉吸收并限制镉从地下到地上转移
30d镉处理后测定了PvBiP2-OE株系和WT中的镉吸收和镉迁移。结果表明,50~100μmol·L-1CdCl2处理下,PvBiP2-OE株系地上部分茎中镉浓度分别比WT中低33.3%~56.1%和7.1%~22.1%(图9A)。相反,根系中则分别高到7.1%~15.4%和25.2%~42.2%(图9B),引起的结果是从根到茎中的镉迁移系数分别低了42.1~59.3%和35.0%`38.2%(图9D)。值得注意,50~100μmol·L-1CdCl2处理下单个植株中镉含量PvBiP2-OE株系中显著高于WT中(图9C)。上述结果说明过表达PvBiP2促进了镉吸收,但能限制镉从根系向地上转移,从而缓解镉对茎叶生长的胁迫。
Claims (3)
1.一种具有提高镉抗性及促进生物量提升的植物编码基因,CDS序列如seq.id.no.1所示。
2.一种由权利要求1所述基因编码的蛋白,序列如seq.id.no.2所示。
3.一种由权利要求1所述基因构建的pCambia1305.2-PvBiP2转基因株系。
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